Analgesic peptides from venom of Grammostola spatulata and use thereof

ABSTRACT

The present invention provides novel methods of treating pain comprising administering to a mammal in need of such treatment an effective analgesic amount of a peptide having the amino acid sequence of SEQ ID. NO.: 1 or SEQ ID NO: 2. The invention further provides a purified peptide having the amino acid sequence of SEQ ID NO: 1. The peptides of SEQ ID NO.: 1 and SEQ ID NO.: 2 can also be used in methods for identifying compounds having analgesia-inducing activity.

This is a division of co-pending application Ser. No. 09/018,799 filedon Feb. 4, 1998 which in turn is a division of application Ser. No.08/775,476 filed on Dec. 30, 1996, now U.S. Pat. No. 5,776,896. Thisapplication claims the benefit of U.S. Provisional Application No.60/009,581 filed on Jan. 3, 1996.

This application claims priority of Provisional Application Ser. No.60/009,581 which was filed Jan. 3, 1996.

FIELD OF THE INVENTION

The present invention relates to peptides that induce analgesia inmammals. More particularly, the present invention relates toanalgesia-inducing peptides obtainable from venom of Grammostolaspatulata, the Chilean pink tarantula spider.

BACKGROUND OF THE INVENTION

Pain is one of the basic clinical symptoms seen by every physician andis usually categorized into three segments: mild, moderate and severe.The mild-to-moderate segment has multiple product entries includingaspirin, acetaminophen, ibuprofen, and other non-steroidal,anti-inflammatory (NSAID) products. Narcotic analgesics remain themainstay of currently marketed products for the treatment ofmoderate-to-severe pain.

Cancer and the post-operative surgical period are two conditions mostoften associated with moderate-to-severe pain. Tumor infiltration ofbone, nerve, soft tissue or viscera are the most common causes of cancerpain accounting for 65-75% of patients. Pain as a result of cancertreatment from surgery, chemotherapy or radiation accounts for 15-25% ofpatients, with the remaining 5 -10% reporting pain independent of theircancer or cancer therapy. Various factors influence the prevalence ofcancer pain including the primary tumor type, stage and site of diseaseand patient variables, especially psychological variables. Similarly,patient response to post surgical pain is dependent upon location andextent of intervention as well as personal attributes. However, postsurgical pain is distinguished from cancer pain by length of treatmentperiod.

The major concern with narcotics, which constitute the largest segmentof the U.S. market for treatment of moderate-to-severe pain, is thepotential for addiction and loss of activity (i.e. tolerance) withcontinued use. Consequently, there is a need for other analgesics thatcan relieve pain, especially moderate-to-severe pain associated withcancer. In order to improve analgesic responsiveness and reduce sideeffects, research efforts have focused on both drug delivery strategiesand novel drug entities. Newer drug delivery strategies includetransdermal narcotics, PCA, intraspinal implantation of controlledrelease pumps and implantation of encapsulated living cells whichrelease naturally-occurring endorphins or other analgesic peptides. Newdrug approaches reflect the varying pathways and causes of moderate-tosevere pain. Classes of compounds in development for treating paininclude serotonergics, noradrenergics, opioid partial agonists and kappaopioid agonists. Therapeutic targets with significant preclinicalinvestigation include tachykinin/bradykinin antagonists and excitatoryamino acid antagonists. Newer targets being exploited include growthfactors, cytokines, nitride oxide regulators, etc. Natural sourcesincluding folk medicine remedies and frog venom extracts are also underinvestigation.

Investigations of spider venoms for identification of biologicalentities with commercial potential has focused primarily on theagrochemical sector. The ultimate goal of these activities has been thesearch for chemical constituents which interact selectively withinvertebrate species to induce paralysis and/or death with minimalmammalian toxicological properties. However, in recent years, spidervenoms have joined other predator-derived venoms being exploited foridentification of compounds which identify mammalian targets and whichassist the development of pharmaceuticals. The arachnid speciesGrammostola spatulata, commonly referred to as the Chilean pinktarantula spider, is a member of the Theraphosidae family and theChelicerata order. Previous studies by Lampe et al. (1993) MolecularPharmacology 4:451-460 showed that venom of Grammostola spatulatacontains a peptide which interacts in a non-selective manner withvoltage-sensitive calcium channels.

SUMMARY OF THE INVENTION

The present invention provides methods of treating pain comprisingadministering to a mammal in need of such treatment an effectiveanalgesic amount of a peptide having the amino acid sequence

Tyr-Cys-Gln-Lys-Trp-Leu-Trp-Thr-Cys-Asp-Ser-Glu-Arg-Lys-Cys-Cys-Glu-Asp-Met-Val-Cys-Arg-Leu-Trp-Cys-Lys-Lys-Arg-Leu-NH2(referred to herein as GsAF I) (SEQ ID NO:1)

or

Tyr-Cys-Gln-Lys-Trp-Met-Trp-Thr-Cys-Asp-Glu-Glu-Arg-Lys-Cys-Cys-Glu-Gly-Leu-Val-Cys-Arg-Leu-Trp-Cys-Lys-Lys-Lys-Ue-Glu-Trp(referred to herein as GsAF II) (SEQ ID NO:2).

An additional aspect of the invention provides a purified peptide havingthe amino acid sequence

Tyr-Cys-Gln-Lys-Trp-Leu-Trp-Thr-Cys-Asp-Ser-Glu-Arg-Lys-Cys-Cys-Glu-Asp-Met-Val-Cys-Arg-Leu-Trp-Cys-Lys-Lys-Arg-Leu-NH2(GsAF I) (SEQ ID NO: 1)

A further aspect of the present invention provides pharmaceuticalcompositions comprising a pharmaceutically acceptable carrier or diluentand a peptide having the amino acid sequence

Tyr-Cys-Gln-Lys-Trp-Leu-Trp-Thr-Cys-Asp-Ser-Glu-Arg-Lys-Cys-Cys-Glu-Asp-Met-Val-Cys-Arg-Leu-Trp-Cys-Lys-Lys-Arg-Leu-NH2(SEQ ID NO: 1)

An additional aspect of the invention provides methods for identifyingcompounds that mimic the analgesia-inducing activity of GsAF I and/orGsAF II.

The present invention additionally provides antibodies specific for GsAFI. The antibodies can be monoclonal or polyclonal. Antibodies can beprepared using methods known in the art such as the methods in Harlow etal. eds., Antibodies: A Laboratory Manual, New York, cold Spring HarborLaboratory Press (1988).

DETAILED DESCRIPTION OF THE INVENTION

Applicant has discovered that peptides from venom of the Chilean pinktarantula spider, Grammostola spatulata, have analgesia-inducingproperties and are thus useful as analgesics for treatment of pain inmammals, including humans, and as research tools for identification ofcompounds that mimic the analgesic activity of the peptides.

Thus, the present invention provides a method for treating paincomprising administering to a mammal in need of such treatment aneffective analgesic amount of a peptide having the amino acid sequence

Tyr-Cys-Gln-Lys-Trp-Leu-Trp-Thr-Cys-Asp-Ser-Glu-Arg-Lys-Cys-Cys-Glu-Asp-Met-Val-Cys-Arg-Leu-Trp-Cys-Lys-Lys-Arg-Leu-NH2(GSAF I) (SEQ ID NO:1) ; or

Tyr-Cys-Gln-Lys-Trp-Met-Trp-Thr-Cys-Asp-Glu-Glu-Arg-Lys-Cys-Cys-Glu-Gly-Leu-Val-Cys-Arg-Leu-Trp-Cys-Lys-Lys-Lys-Ile-Glu-Trp(GsAF II) (SEQ ID NO:2)

The peptides are useful for treating pain in mammals, including humans,conventional laboratory animals such as rats, mice and guinea pigs,domestic animals such as cats, dogs and horses, and any other species ofmammal. The peptides can be used to treat acute or chronic pain from anysource or condition, such as burns, cancer, neuropathies, organinflammation or surgical intervention. Preferably, however, the peptidesare used to treat moderate-to-severe pain due to cancer or surgery. Thepeptides can be administered orally, parenterally, intrathecally,topically, intraveneously, intramuscularly or intradermally/epineurally.A preferred route of administration is intrathecally.

The peptides thereof can be prepared for pharmaceutical use byincorporating them with a pharmaceutically acceptable carrier ordiluent. Thus, a further aspect of the present invention providespharmaceutical compositions comprising a peptide from Grammostolaspatulata as described herein and a pharmaceutically acceptable carrieror diluent. The peptide can be prepared for pharmaceutical use byincorporating it in unit dosage form as tablets or capsules for oral orparenteral administration either alone or in combination with suitablecarriers such as calcium carbonate, starch, lactose, talc, magnesiumstearate, and gum acacia. The peptide can be formulated for oral,parenteral or topical administration in aqueous solutions, aqueousalcohol, glycol or oil solutions or oil-water emulsions.Buffered-aqueous or carrier mediated aqueous/non-aqueous intrathecal andintraveneous dosages can be formulated. These and other suitable formsfor the pharmaceutical compositions of the invention can be found, forexample, in Remington's Pharmaceutical Science, 15th ed., MackPublishing Company, Easton, Pa. (1980). The pharmaceutical compositionsof the invention can comprise any combination of one or both of thepeptides.

The amount of the active component (i.e. peptide) in the pharmaceuticalcompositions can be varied so that a suitable dose is obtained and aneffective analgesic amount can be administered to the patient. Thedosage administered to a particular patient will depend on a number offactors such as the route of administration, the duration of treatment,the size and physical condition of the patient, the potency of thepeptide and the patient's response thereto. An effective analgesicamount of the peptide when administered intrathecally is generally inthe range of from about 5 nanograms per kilogram body weight of thepatient to about 500 micrograms per kilogram; preferably from about 50nanograms per kilogram to about 50 micrograms per kilogram; morepreferably from about 500 nanograms per kilogram to about 5 microgramsper kilogram. Effective amounts of the peptide will vary whenadministered by other routes. An effective analgesic amount can beestimated by testing the peptide in one or more of the pain testsdisclosed herein to arrive at a dose that can be varied according to oneor more of the criteria listed above to provide a suitable amount of thepeptide to the mammal.

The terms"inducing analgesia", "analgesia-inducing activity","analgesia-producing" and similar terms refer to the ability of thepeptide to treat pain in mammals or attenuate pain as evidenced byfavorable results in one or more conventional laboratory models fortesting pain or assessing analgesia such as the tests set forth herein.

Analgesic activity of the peptides is determined by testing in at leastone, and preferably more than one, of a series of tests whichincludes 1) tail flick latency (Abbott, F. V. et al., Pharmacol.Biochem. Behav., 17, 1213-1219, 1982; Cridland, R. A. and Henry, J. L.,Brain Res., 584:1-2, 163-168, 1992), 2) hot plate threshold (Woolfe, Gand Macdonald, A. A., JPET, 80, 300, 1944; Ankier, S. I., European J.Pharmacol., 27, 1-4, 1974), and 3) vonFrey filament threshold (Kim, S.H. et al., Pain, 55, 85-92, 1993).

The tail flick latency and hot plate threshold tests are measurements ofthermal nociception. The von Frey filament threshold test evaluatesmechanical nociceptive activity. All three of these pain tests evaluatethe analgesic activity of compounds against the phasic stimulation ofeither thermal- or mechanical-nociceptors and reflect to a large degreethe activation of A- and polymodal C-fiber afferents. Clinicalanalgesics with an opioid-based mechanism of activity are efficacious inthese tests, whereas those analgesics which either interactpreferentially with peripheral targets or possess multiple sites ofaction are generally less active. These tests are good predictors ofmoderate to strong analgesic agents and within the opioid class ofcompounds the correlation with clinical effect is good. Thenon-steroidal anti-inflammatory (NSAID) class of analgesics, whichclinically target the lower end of the pain scale, are not routinelydetected under the parameters normally used for these tests.

Analgesic detection of NSAIDs is dependent upon the generation of anociceptive status of increased responsiveness (i.e. a lowering ofthreshold to noxious stimuli) in response to primary afferent tissuedamage and inflammation. Interaction between the immune and nervoussystems to induce this state represents the target for NSAID activity.Inhibition of this heightened activity of peripheral nociceptors, and ofthe corresponding central circuitry, is detected over longer timeintervals by either monitoring spontaneous behavior or the response tosubsequent noxious stimuli. These more chronic measurements of the"hyperalgesic" status are considered to mimic most clinical conditionsof pain. They also broaden the detection capability for useful analgesicagents without exclusion of active agents detected in the phasic paintests. Numerous pain tests have been developed to model this conditionin laboratory animals. The noxious stimuli used to induce this conditionare either chemical irritants/caustic agents or inflammatorystimulators. Within these tests, the major defining variable is the timeinterval required for the development, and the ethically justifiableduration, of the hyperalgesic/inflammatory state. Compounds can beevaluated for their intrinsic activity to prevent the development of thehyperalgesic condition (i.e. compound administered prior to noxiousstimulant) or to reduce the increased nociceptive response (i.e.compound administered post-noxious stimulation) or both. Primary endpoints in these tests are measurements of nociceptive and inflammatorystatus.

The formalin test (Dubuisson, D. and Dennis, S. G., Pain, 4, 161-174,1977) was used since it produces a well delineated bi-phasic responsethat is considered to be indicative of tonic versus acute pain and canbe performed within a reasonably short time period (i.e. <1 hr). Theinitial phase of this response is triggered by a substantial primaryafferent barrage, similar in character to that described for the acutephasic tests except that chemical nociceptors are the mediators. Thesecond phase is considered to be the hyperalgesic spontaneous activitythat results from the initial tissue damage and reflects the lowering ofthe nociceptive threshold plus the priming or "wind up" of thecorresponding spinal circuitry. Hence, both peripheral and centralneuronal circuits and mediators are required to induce and sustain thispainful tissue-injury condition.

The formalin model in rodents has been validated as a predictive test oftreating injury-induced pain in humans (Dennis, S. G. and Melzack, R.,In: Advances in Pain Research and Therapy, vol. 3, 747-759, Eds. J. J.Bonica et al., Raven Press:New York, 1979; Tjolsen, A. et al., Pain, 51:5-17, 1992.). Evaluation of clinically used analgesics in this model hasconsistently demonstrated a strong correlation with human efficacy foropioid based compounds or drugs known to interact with opioid systems(Wheeler-Aceto, H., "Characterization Of Nocioception And Edema AfterFormalin-Induced Tissue Injury In The Rat: Pharmacological Analysis OfOpioid Activity", Doctoral Dissertation, Temple School of Medicine,Philadelphia, Pa., 1994; Shibata, M. et al., 38, 347-352, 1989).Efficacy and potency profiles for milder analgesic drugs possessingprimarily NSAID based mechanisms of action have produced equivocalresults (Wheeler-Aceto, H., Doctoral Dissertation, Temple School ofMedicine, Philadelphia, Pa., 1994; Hunskaar, S. et al., Neurosci. Meth.,14, 69-76, 1985; Shibata, M. et al., Pain, 38, 347-352, 1989; Malmberg,A. B. and Yaksh, T. L., J. Pharmacol. Exp. Ther., 263, 136-146, 1992).These equivocal findings in the formalin model reflect experimentaldifferences in how the test is conducted such as the parameters of thetest (i.e. stimulus intensity administered, response measurement andresponse interval analyzed), species and strain of laboratory animalused and route/timing of administration of compounds (for review,Wheeler-Aceto, H., Doctoral Dissertation, Temple School of Medicine,Philadelphia, Pa., 1994). However, consensus exists that high efficacyanalgesics used to treat moderate to severe pain are detected in thistest independent of these experimental differences. If these compoundshave limited central nervous system penetration, less activity isdetected.

In addition to their use as analgesics, the peptides are useful inbiological assays such as assays to detect compounds that mimic theanalgesic activity of the peptides, assays to detect the anatomical siteof action of the peptides, or studies on the mechanism of action of thepeptides. Thus, another aspect of the invention provides methods fordetecting compounds that mimic the analgesic activity of GsAF I and/orGsAF H. Mimicking the activity of the peptides disclosed herein refersto the ability of test compounds to induce analgesia, bind to cellularreceptors to which the peptides bind or otherwise act in the same orsimilar physiological manner as the peptides. Thus, the presentinvention provides methods for identifying compounds havinganalgesia-inducing activity or which otherwise mimic the activity ofGsAF I and/or GsAF II comprising the steps of adding a test compound toa biological assay that determines activity of a peptide having theamino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; and detecting theactivity of the test compound.

Biological assays to identify compounds that mimic the activity of GsAFI and/or II can be in vivo assays, such as those described herein, or invitro assays, such as the assays described below. For example, GsAF Iand/or GsAF II can be used in competitive binding screening assays toidentify compounds that mimic the activity of GsAF I and II according tothe following method. A test compound and detectably labeled peptide areadded to mammalian cells or tissue under conditions that allow bindingto the cells or tissue. Binding of labeled peptide to the mammaliancells or tissue is then measured. Compounds that mimic the activity ofthe detectably labeled peptide will compete with the peptide for bindingsites on the receptor. Consequently, a smaller amount of detectablelabel will be measured when the test compound mimics the activity of thepeptide by binding to the receptor than when the test compound does notmimic the activity of the peptide and does not bind to the receptor, ordoes so with much less affinity. In particular, GsAF I and/or II couldbe labeled with ¹²⁵ I and used in the assay described in Stumpo et al,Eur. J. Pharmacol. 206:155, 1991 and modified from Abe et al, Neurosci.Lett. 71:203, 1986. Briefly, individual test compounds are preincubatedwith brain or spinal cord membrane tissue prior to the addition of ¹²⁵I-labeled GsAF I and/or II, followed by incubation to allow binding tooccur. The reaction mixture is then filtered and the filters containingthe brain or spinal cord membrane tissue are rinsed with buffer. Bindingof ¹²⁵ I-labeled peptide can be determined by scintillation counting.Compounds that mimic the action of GsAF I and II will compete with thelabeled peptide and produce lower levels of labeled peptide binding tothe receptor on the cells of the brain or spinal cord membrane tissuethan compounds that do not mimic the activity of GsAF I or II.Nonspecific binding will be defined as that remaining in the presence ofexcess (100-1,000×) unlabeled GsAF I or GsAF II.

For use as a reagent in biological assays, the peptides preferablyincorporate a detectable label. The detectable label can be anyconventional type of label and is selected in accordance with the typeof assay to be performed. For example, the detectable label can comprisea radiolabel such as ¹⁴ C, ¹²⁵ I, or ³ H, an enzyme such as peroxidase,alkaline or acid phosphatase, a fluorescent label such asfluoroisothiocyanate (FITC) or rhodamine, an antibody, an antigen, asmall molecule such as biotin, a paramagnetic ion, a latex particle, anelectron dense particle such as ferritin or a light scattering particlesuch as colloidal gold. Suitable method to detect such labels includescintillation counting, autoradiography, fluorescence measurement,calorimetric measurement or light emission measurement. Detectablelabels, procedures for accomplishing such labeling and detection of thelabels are well known in the art and can be found, for example, in AnIntroduction to Radioimmunoassays and Related Techniques: LaboratoryTechniques in Biochemistry and Molecular Biology,4th Ed., T. Chard,Elsevier Science Publishers, Amsterdam, The Netherlands, 1990; Methodsin Non-Radioactive Detection, Gary C. Howard, Ed., Appleton and Lange,East Norwalk, Conn., 1993 or Radioisotopes in Biology: A PracticalApproach, R. J. Slater, Ed., IRL Press at Oxford University Press,Oxford, England, 1990.

Additionally, the peptides can be used in the assay of Keith et al, J.Auton. Pharmacol., 9:243-252, 1989 and Mangano et al, Eur. J. Pharmacol.192:9-17, 1991 to identify compounds that mimic the activity of GsAF Ior II. Briefly, this assay measures K⁺ -evoked release of ³H-D-aspartate and ³ H-norepinephrine from rat brain or spinal cordslices. Spinal cord or brain slices can be pre-equilibrated with GsAFI/GsAF II, test compound or vehicle for 15 min prior to K⁺ stimulation.Levels of K⁺ -induced release of ³ H-norepinephrine and ³ H-D-aspartateare measured. Inhibition of K⁺ -induced release of ³ H-norepinephrineand ³ H-D-aspartate by GsAF I/GsAF or test compound versus inhibitiondue to the vehicle control is then determined. Test compounds can bescreened to determine both absolute inhibitory activity as well asactivity relative to GsAF I/GsAF II.

The compounds that mimic the analgesic activity of GsAF I and/or II willthemselves have analgesic activity and can be used as analgesics or forother purposes such as determining the anatomical site of action,determining the mechanism of action of the peptides and in screeningassays to identify other compounds that mimic the analgesic activity ofthe peptides. Preferably test compounds used in the screening assay aresmall organic molecules but analgesic activity of any type or size ofcompound such as proteins and peptides can also be tested with themethods of the invention.

GsAF I and/or II can be used in assays to identify its site of actionand for further physiological characterization of its activity. Forexample, the peptides can be used to study inhibition ofbinding/interaction of labelled ligand to mammalian tissues, isolatedcells or subcellular components derived therefrom. Similarly, thepeptides can be used to study inhibition of binding/interaction oflabelled ligand to specific recombinantly expressed proteins generatedfollowing either cDNA or genomic transformations/transfections ofeukaryotic or prokaryotic host systems. The peptides can be used tostudy analogous biochemical interaction with mammalian tissue functionto include receptor mediated activation/inhibition of specifiedtransduction pathways, movement of ionic species across biologicalmembranes and alteration of transcriptional/translational profile ofspecific pain-induced gene activity. Specifically, methods to measurethe alteration of potassium, sodium, calcium, chloride or hydrogen ionicdistribution across mammalian cell derived membrane barriers as measuredby either radioisotopic or fluorescent detection of specified ionicspecies can be utilized. The effects of these ionic movements upon theregulation of specific immediate early genes can be studied as well.

The peptides can additionally be used for electrophysiologicalmeasurements of potassium, sodium, calcium and chloride distributionacross mammalian cell membranes to include macroscopic analysis ofsynaptic transmission as well as microscopic analysis of specified ioniccurrents. Specifically, inhibition of noxious-mediated neuronal firingand synaptic transmission within spinal dorsal horn neurons can beanalyzed as well as inhibition of isolated specific ionic currentswithin individual dorsal root ganglion or spinal dorsal horn neurons.

The peptides can further be used in studies of inhibition ofphysiological response to nociofensive/noxious stimuli administered tomammalian species. Specifically, motor parameters (i.e. limb withdrawalthresholds or response time latencies/durations) can be quantitated inresponse to either thermal, mechanical or chemical noxious stimuliadministered to either naive animals or animals in which a painfulcondition has been experimentally induced.

GsAF I and II can be prepared by isolation from Grammostola spatulatavenom, chemical synthesis or recombinant DNA methods. Grammostolaspatulata venom is commercially available from Spider Pharm,Feasterville, Pa., USA. The peptides are preferably isolated from spidervenom by sequential fractionation using reverse phase-high pressureliquid chromatography on C-8 and C-18 silica supports withtrifluoroacetic acid/acetonitrile buffer. A preferred C-8 silica supportis Zorbax® Rx C-8 (Mac-Mod Analytical, Inc., West Chester, Pa.) which iscomprised of 5 micron diameter silica particles having 300 Å pore sizeand covalently modified to contain diisopropyloctyl side chains. TheC-18 silica support is preferably comprised of 5 micron diameter silicaparticles having 300 Å pore size and covalently modified to contain anoctadecyl side chain. Other types of C-8 and C-18 silica supports arealso suitable for use in isolating the peptides. A preferred buffer is0.1% trifluoroacetic acid in acetonitrile. In a preferred method, crudevenom is initially fractionated on a C-8 semi-preparative column using abroad 20-50% gradient of 0.1% trifluoroacetic acid in acetonitrilebuffer. The peptides are further purified using a C-8 column andshallower gradients of the same buffer, followed by additionalfractionation using a C-8 column and the broad buffer gradient.

GsAF I and II can be prepared by recombinant DNA techniques. A DNAsequence coding for one of the peptides is prepared, inserted into anexpression vector and expressed in an appropriate host cell. The peptidethus produced is then purified from the host cells and/or cell culturemedium. Methods for preparing DNA coding for the peptides and expressionof the DNA are well-known and can be found, for example, in Sambrook etal. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,N.Y.: Cold Spring Harbor Laboratory Press, Guide to Molecular CloningTechniques: Methods in Enzymology, vol. 152, S. L. Berger and A. R.Kimmel, Ed., Academic Press (San Diego, Calif.), 1987 and Gene Transferand Expression Protocols: Methods in Molecular Biology, vol. 7, E. J.Murray, Ed., Humana Press (Clifton, N.J.), 1991.

The peptides can also be prepared by chemical synthesis using eitherautomated or manual solid phase synthetic technologies. These techniquesare well known in the art and are differentiated on the basis offeatures such as selection of synthetic resin backbone, selection ofamino, carboxyl and side chain protecting groups and selection ofdeprotection strategies. Methods for synthesizing peptides can be foundin standard texts such as E. Atherton and R. C. Sheppard, Solid PhasePeptide Synthesis: A Practical Approach, IRL Press/Oxford UniversityPress, Oxford, UK, 1989 and M. Bodanszky, Peptide Chemistry: A PracticalTextbook, Springer-Verlag, New York, USA, 1988.

In a preferred synthetic method, synthesis of GsAF I and GsAF II can bedone using Fmoc chemistry on an automated synthesizer. Dependent uponquantitative yields, production of the linear reduced peptide can beperformed in either a single process or in two different processesfollowed by a condensation reaction to join the fragments. A variety ofprotecting groups can be incorporated into the synthesis of linearpeptide to facilitate isolation, purification, and/or yield of thedesired peptide. Protection of cysteine residues found in the peptidecan be accomplished using, for example, a triphenylmethyl,acetamidomethyl and/or 4-methoxybenzyl group in any combination. Such astrategy may offer advantages for subsequent oxidation studies to yieldfolded peptide. Differential proteolytic digestion of native GsAF I andGsAF II coupled to mass spectrometric analysis of the resultantfragments can be utilized for assignment of intramolecular disulfidebonds. This data can be subsequently incorporated into synthetic peptidestrategies to increase yields. Oxidative strategies include random airoxidation, iodine assisted oxidation, and dimethylsulfoxide assistedoxidation, as well as the use of small quantities of thiol reagents inan oxidation reaction to attain the desired folded peptide. Crude,linear, reduced peptides, as well as homogeneous, oxidized peptides, canbe purified using reverse-phase high pressure liquid chromatography HPLC(RP-HPLC) or other standard techniques.

A further aspect of the invention provides a novel peptide which has theamino acid sequence

Tyr-Cys-Gln-Lys-Trp-Leu-Trp-Thr-Cys-Asp-Ser-Glu-Arg-Lys-Cys-Cys-Glu-Asp-Met-Val-Cys-Arg-Leu-Trp-Cys-Lys-Lys-Arg-Leu-NH2(GsAF I) (SEQ ID NO: 1)

The leucine at the carboxy terminus of the peptide is amidated, i.e.,the free end of the terminal leucine residue ends with --CO--NH₂ insteadof --COOH. Both amidated and non-amidated peptides are within the scopeof the present invention.

As used herein, a purified or isolated peptide refers to a peptide thatis substantially free of contaminating cellular components, other venomconstituents or other material such as reagents used in chemicalsynthesis of the peptide. Preferably, the peptide is present in amixture containing the peptide in an amount greater than about 50% ofthe total mixture, more preferably in an amount greater than about 80%,most preferably in an amount greater than about 90%.

EXAMPLES Example 1--Isolation and Characterization of Peptide GsAF Ifrom Venom of Grammostola spatulata

A. Isolation of Peptide

Crude Grammostola spatulata venom was supplied as frozen aliquots bySpider Pharm, Inc. (Feasterville, Pa. 19053). Reverse phase-highpressure liquid chromatography (RP-HPLC) of the venom was performedusing C-8 semi-preparative (25 cm×9.4 mm) and analytical (25 cm×4.6 mm)columns (Zorbax® RX-C8, Mac-Mod Analytical, Inc. West Chester, Pa.,which is comprised of 5 micron silica microsphere particles having a 300Å pore size and covalently modified with diisopropyl octyl side chains);and a C-18 analytical (25 cm.×4.6 mm) column (Vydac, Hesperia, Calif.,which is comprised of 5 micron silica microsphere particles having a 300Å pore size and covalently modified with octadecyl side chains).Semi-preparative scale RP-HPLC was done using a five milliliter perminute flow rate whereas a one milliliter per minute flow rate was usedfor the analytical analyses.

Detection of eluting entities was monitored via ultraviolet spectroscopyat 215 nm and fractions were either collected at 1 minute intervals ormanually based upon ultraviolet intensity. Initial injection volumes of30-50 microliter crude venom were made. Consequently, multiplefractionations were carried out at each stage of the purification withpooling of individually identical fractions. All fractions werelyophilized prior to resuspension in HPLC grade H₂ for subsequentpurification or in vivo analgesic testing. Resuspension volumes werebased upon original crude venom volumes. Analgesic evaluation was doneon samples deemed to be greater than 90% homogeneous by RP-HPLC. Sampleswere stored at 4° C. following resuspension. No detectable loss ofactivity was witnessed with storage or with adherence to either plasticor glass.

Initial fractionation of crude Grammostola spatulata venom on theZorbax® RX-C8 semipreparative column was done with a 20-50% gradient ofTFA/CH₃ CN Buffer (0.1 % trifluoroacetic acid in acetonitrile) over 30minutes with a 3 minute delay. (TFA/CH₃ CN Buffer was prepared by adding4 ml of trifluoroacetic acid to 4 liters of acetonitrile.) Column flowrate was 5 milliliters per minute and fractions collected at one minuteintervals. Fraction 18 was highly enriched for GsAF I. Fraction 17 alsocontained GsAF I peptide but in smaller quantities than fraction 18 withmost purifications. Following lyophilization and resuspension offraction 18, and optionally fraction 17, further separations wereperformed with shallower gradients of TFA/CH₃ CN Buffer.

Fraction 18 (and optionally 17) were applied to a Zorbax® RX-C8semi-preparative column and fractionated using a 24-30% gradient ofTFA/CH₃ CN Buffer over 24 minutes, with 3 minute delay. The major UVabsorbing peak was manually collected with removal of peak tails. Afterthis step, sample purity was usually found to be at least 85%.

The major UV absorbing peak collected in the previous step was furtherpurified using a 20 -50% gradient of TFA/CH₃ CN Buffer on a Zorbax®(RX-C8 semi-preparative column (flow rate 5 mil/min) over 30 min with a3 minute delay. The primary peak which elutes at 22 minutes wascollected manually with removal of peak tails. GsAF I sample purity wasfound to be about 98% pure.

On occasion, exposure of the GsAF I sample to a very shallow gradient of48-51% TFA/CH₃ OH buffer on a Zorbax® RX-C8 semi-preparative column over21 minutes, followed by lyophilization, resulted in the appearance oftwo RP-HPLC resolvable peptides that differ in mass by 16 Daltons. Frominternal studies done with another peptide sample, this massdifferential does not translate into a differential primary amino acidsequence but most likely reflects a side-chain adduct.

B. Characterization of Peptide

1. Electrospray Mass Spectrometry (ES-MS) Analysis of Molecular Weightand Disulfide Bridge Assignment:

Electrospray spectra were acquired for the peptide using a massspectrometer (VG/Fisons QUATTRO, Fisons Instruments, Inc. Manchester,UK) in the continuum acquisition mode. The (M+3H)³⁺, (M+4H)⁴⁺ and(M+5H)⁵⁺ charge states were observed for each sample and mathematicallytransformed to yield a zero charge state spectrum. Analyses wereperformed on both the native/oxidized and the reduced state of thepeptide. Lyophilized GsAF I was reduced in 0.5M dithio-threitol (DTT)0.1M N-ethylmorpholine, pH 8.5, at 38° C. for 10 min. Flow injectionscontaining approximately 200-400 picomoles of peptide were measured. Theaverage molecular weight of GsAF I was determined to be 3707.5 Daltons(Da). After thiol reduction, the average molecular weight was measuredat 3713.5 Daltons. Since each reduction of a disulfide bond increasesthe mass of a peptide by 2 Da, the peptides contain three disulfidelinkages based upon the 6 Da mass shift.

The native oxidized peptide was digested with a combination of modifiedtrypsin (Boehringer Mannheim) and endoproteinase Asp-N proteases. Theresulting mixture of proteolysis products was subjected to liquidchromatography-electrospray mass spectral analysis to assign disulfidelinkage. Multiple peptides containing a disulfide bridge linking aminoacids 9 and 21 of the GsAF I peptide were observed. Scrambling ofdisulfide bonds may occur when proteolysis is performed at pH 8;however, randomization of proteolysis products was not observed, anddisulfide bond scrambling is thought to be unlikely in this case. Linksfor the remaining disulfide bridges were not established. Subsequentanalysis of the crude proteolysis mixture by matrix-assisted laserdesorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (VGAnalytical/Fisons TOFSpec-SE, Fisons Instruments, Inc. Manchester, UK)provided confirmation of the electrospray mass spectral measurements.

2. N-terminal Sequence Analysis of Reduced, Pyridylethylated NativePeptides and Proteolytically Digested Fragments:

N-terminal sequencing was performed on a gas phase sequencer (AppliedBiosystems 475, Foster City, Calif.). SDS-Page was performed using a16.5% high cross linked Tris-Tricine gel (Schagger, H. and G. von Jagow,Anal. Biochem. 166:368-379, 1987) and electroblotted to ProBlot (AppliedBiosystems, Foster City, Calif.)) as described by Matsuidara, P., J.Biol. Chem. 262:10035-10038. Electroblotted bands were pyridylethylatedin the gas phase according to the method described in Andrews, P.C. andJ. E. Dixon, Anal. Biochem. 161:524-528, 1987. Covalent attachment ofpeptides via activation of carboxyl groups and reaction with arylaminederivatized polyvinylidene difluoride using sequalon membranes(Millipore, Inc., Milford, Mass.) was performed according to themanufacturer's instructions. V8 proteolytic digestion of reduced(100×dithiothreitol vs. Cys on mole basis) GsAF I peptide was done in 50mM Na phosphate buffer, pH 7.8, for 18 hr. using an enzyme:substrateratio of 1:44. Fragments were isolated using RP-HPLC and their massanalyzed using laser desorption/ionization mass spectrometry prior tosequence analysis. Samples were applied to the sequencer either asdirect solutions onto a coated disc or as covalent coupled entities toascertain carboxyl terminal acidification/amidation. Shown below is thesequence obtained for peptide GsAF I. Amidation of the GsAF I peptide issupported by the ES-MS data for the intact, native peptide and for therespective V8 (or tryptic as well) carboxyl terminal fragment.

The sequence obtained for peptide GsAF I is

Tyr-Cys-Gln-Lys-Trp-Leu-Trp-Thr-Cys-Asp-Ser-Glu-Arg-Lys-Cys-Cys-Glu-Asp-Met-Val-Cys-Arg-Leu-Trp-Cys-Lys-Lys-Arg-Leu-NH2(SEQ ID NO:1)

Leu-NH2 denotes that the terminal leucine residue is amidated, i.e., thefree end of the terminal leucine residue ends with --C(═O)--NH₂ insteadof --COOH. The amino acid sequence of the peptide is presented startingwith the amino terminus.

3. UV Spectroscopy:

A complete spectrum was obtained for the peptides using a 8452A diodearray spectrophotometer (Hewlett Packard, Avondale, Pa., USA).Concentrations of the final peptides were deduced from the Abs_(280nm).Based upon the differential contributions from 3 Trp, 1 Tyr and 6 Cys,the calculated molar extinction coefficient of GsAF I at 280 nm is18710. In cases where sufficient peptide was isolated for accurate massweighing (and assuming appropriate peptide content as a result of theTFA salt), the respective concentration values were in good agreement.Using either method of quantitation, and multiple preparations of nativeGsAF I, the venom concentration of GsAF I is estimated to beapproximately 500-750 μM.

Example 2--Isolation and Characterization of Peptide GsAF II

A. Isolation of Peptide

Crude Grammostola spatulata venom was supplied, as frozen aliquots, bythe commercial vendor Spider Pharm, Inc. (Feasterville, Pa. 19053).Reverse phase-high pressure liquid chromatography (RP-HPLC) of the venomwas performed using Zorbax® Rx-C8 semi-preparative (25 cm×9.4 mm) andanalytical (25 cm×4.6 mm) columns (Mac-Mod Analytical, Inc. WestChester, Pa.; Zorbax® Rx-C8 is comprised of 5 micron silica microsphereparticles having a 300 Å pore size and covalently modified withdiisopropyl octyl side chains) and a C-18 analytical (25 cm×4.6 mm)column (Vydac, Hesperia, Calif.; the C-18 support is comprised of 5micron silica microsphere particles having a 300 Å pore size andcovalently modified with octadecyl side chains). Semi-preparative scaleRP-HPLC was done using a 5 milliliter/minute flow rate whereas a 1milliliter per minute flow rate was used for the analytical analyses.

Detection of eluting entities were monitored via ultraviolet (UV)spectroscopy at 215 nm and fractions were either collected at 1 minuteintervals or manually based upon UV intensity. Initial injection volumesof 30-50 microliter (μl) crude venom were made. Consequently, multiplefractionations were carried out at each stage of the purification withpooling of individually identical fractions. All fractions werelyophilized prior to resuspension in HPLC grade H₂ O for subsequentpurification or in vitro testing. Resuspension volumes were based uponoriginal crude venom volumes. Evaluation was done on samples deemed tobe greater than 90% homogeneous by RP-HPLC. Samples were stored at 4° C.following resuspension. No detectable loss of activity was witnessedwith storage or with adherence to either plastic or glass.

Initial fractionation of crude Grammostola spatulata venom on theZorbax® RX-C8 semi-preparative column was done with a 20-50% gradient ofTFA/CH₃ CN Buffer (0.1% trifluoroacetic acid in acetonitrile) over 30min with a 3 minute delay. (TFA/CH₃ CN Buffer was prepared by adding 4ml of trifluoroacetic acid to 4 liters of acetonitrile). Column flowrate was 5 milliliters per minute and fractions collected at one minuteintervals. Fraction 19 was highly enriched for GsAF II. Followinglyophilization and resuspension of fraction 19, further separations ofthis fraction were performed with shallower gradients of TFA/CH₃ CNBuffer.

Fraction 19 was applied to a Zorbax® RX-C8 semi-preparative column andfractionated using either a 29-33% or a 30-34% gradient of TFA/CH₃ CNBuffer over 24 minutes with a 3 minute delay. The major UV absorbingpeak was manually collected with removal of peak tails. After this step,sample purity was usually found to be at least 85%. The major UVabsorbing peak was further purified using a 20-50% gradient of TFA/CH₃CN Buffer over 30 min with a 3 minute delay. The primary peak whichelutes at 23.5 minutes was collected manually with removal of peaktails. GsAF II sample purity was found to be about 98% pure.

B. Characterization of Peptide

The peptide GsAF II was characterized using the methods described forpeptide GsAF I in Example 1. The average molecular weight of GsAF II wasdetermined to be 3979.9 Daltons (Da). After thiol reduction, the averagemolecular weight was 3985.9 Da. Since each reduction of a disulfide bondincreases the mass of a peptide by 2 Da, the peptide contains threedisulfide linkages based upon the 6 Da mass shift.

Amino acid composition analyses were performed using an amino acidanalyzer (Applied Biosystems 420H, Foster City, Calif.). Datanormalization was done with respect to leucine. No discrepancies(excluding those residues which are either partially or totallydestroyed during hydrolysis) in residue/mol values were recorded withrespect to the Edman N-terminal sequencing analysis.

Amino acid composition analysis yielded the data presented in the tablebelow. Since tryptophan is completely destroyed and cysteine ispartially destroyed in this analysis, their presence was inferred fromUV spectroscopy and electrospray mass spectral analysis, respectively.Residue/mol values were calculated on the basis of using Leu as thestandard.

    ______________________________________    Residue     Total Amount (pmole)                              Residue/mol    ______________________________________    Asp/Asn     701.2         1.2    Glu/Gln     2767.6        4.7    Ser         108.9         0.2    Gly         618.1         1.0    His         0             --    Arg         1050.0        1.8    Thr         518.9         0.9    Ala         35.5          0.1    Pro         36.7          0.1    Tyr         547.5         0.9    Val         523.9         0.9    Met         875.7         1.5    Cys         2124.1        3.6    Ile         545.3         0.9    Leu         1186.1        2.0    Phe         48.5          0.1    Lys         2639.7        4.5    ______________________________________

Shown below is the amino acid sequence for the peptide GsAF II:

Tyr-Cys-Gln-Lys-Trp-Met-Trp-Thr-Cys-Asp-Glu-Glu-Arg-Lys-Cys-Cys-Glu-Gly-Leu-Val-Cys-Arg-Leu-Trp-Cys-Lys-Lys-Lys-Ile-Glu-Trp(SEQ ID NO:2)

Deduction of Trp at position 31 of GsAF II is based upon amino acidcompositional data and ES-MS analysis. Specifically, the unaccountedmass difference between the calculated mass value for the Edman deducedsequence and the mass spectral analysis for the native peptide is 186 Daassuming a free acid carboxyl terminus or 187 Da if the carboxylterminus is amidated. This mass difference (+or -1 Da) could beaccounted for by multiple amino acid combinations. However, none ofthose combinations are in good agreement with the amino acid compositiondata. Since the mass of an internal Trp is 186 Da, and Trp is destroyedunder the hydrolysis conditions, assignment of Trp to position 31 as afree acid was tentatively made. This assignment was subsequentlysupported by analysis of the carboxyl fragment of GsAF II isolatedfollowing tryptic digestion. Both high resolution mass spectral analysisand MS-MS sequencing analysis of the fragment demonstrate the presenceof a free acid Trp at the carboxyl terminus. These data were furthercorroborated when identical analyses were obtained for a syntheticallyprepared Lys-Ile-Glu-Trp peptide. Additionally, the RP-HPLC retentionprofile of both the native fragment and the synthetic fragment wereidentical.

Based upon the presence of 4 Trp, 1 Tyr and slight contribution from 6Cys residues, a molar extinction coefficient of 24310 at 280 nm wasdeduced for GsAF II. Using this value , UV spectroscopy analyses ofnative GsAF II preparations indicate that the venom concentration ofthis peptide is approximately 3-5 mM.

Example 3--Analgesic Evaluation--Tail Flick Latency

This test measures the time interval required for a rat to withdraw itstail, via a spinally mediated reflex mechanism, from a high intensitylight source (IITC Inc./Life Sciences Instruments, Woodland Hills,Calif. 91367) focally applied to the dorsal surface of the appendage.The intensity of the light beam has been experimentally defined suchthat naive animals will withdraw their tails within 2 to 4 seconds. Amaximum cut off time for the light source has been set at ten seconds toreduce the amount of secondary tissue damage.

Data is expressed either as absolute time or a percentage of the maximalpossible effect (MPE) which is described by the following equation where10 seconds is the maximum: ##EQU1## (Latency refers to the amount oftime before the animal removed its appendage from the light source.)

GsAF I Administration:

With minimal restraint, peptide GsAF I was injected intrathecally(i.th.) into young (75-150 gram) male Sprague-Dawley rats (CharlesRivers Laboratories, Wilmington, Mass. 01887). I.th. injections weremade into the spinal subarachnoid space between lumbar spinous processesL4 and L5 using 10 microliter Hamilton syringes equipped with 3/8 inchby 28G needles. Dosing levels were based upon concentrations deducedfrom ultraviolet absorbance values at 280 nm using an extinctioncoefficient of 18710. Injection volume was 10 microliters. The injectionvehicle was saline or 0.1% bovine serum albumin(BSA)/saline. The ratswere pretreated with GsAF I 30 minutes prior to exposure to the lightsource.

Complete inhibition of the tail flick response (i.e., latency valuegreater than 10 seconds) was recorded in most rats followingadministration of 180 picomoles (666 nanograms) of GsAF I. A 95% MPE wasattained for this dose and confounding side effects such as motordisturbances, limb impairment/paralysis, righting reflex, sedation,etc.) were either minimal or not present. Logarithmic decreases in thedose resulted in rapid loss of effect. 18 picomoles (66 nanograms) ofGsAF I produced 29% MPE and 1.8 picomoles (6.6 nanograms) was inactive.Maximal activity was detected with a 30 minute pretreatment time.

GsAF II Administration:

With minimal restraint, peptide GsAF II was injected intrathecally(i.th.) into young (75-150 gram) male Sprague-Dawley rats (CharlesRivers Laboratories, Wilmington, Mass.)) into the spinal subarachnoidspace between lumbar spinous processes L4 and L5 using 10 microliterHamilton syringes equipped with 3/8 inch by 28G needles. Dosing levelswere based upon concentrations deduced from ultraviolet absorbancevalues at 280 nm using the deduced molar extinction coefficient of24310. Injection volume was 10 microliters. The injection vehicle wassaline or 0.1% bovine serum albumin(BSA)/saline. The rats werepretreated with GsAF II 15 minutes prior to exposure to the lightsource.

Complete inhibition of the tail flick response (i.e. latency greaterthan ten seconds) was recorded with all animals (n=8) dosed with 2.33nanomoles (9.27 micrograms) of GsAF II. When animals were dosed with 583picomoles (2.33 micrograms) of GsAF II, 100% MPE was recorded for fiveof six animals, with the average MPE at this dose of 92%. Similar toGsAF I, no confounding side effects were detected at these doses.

Example 4--Analgesic Evaluation--Hot Plate Threshold

This test measures the temperature at which point a rat voluntarilyremoves one of its hindlimbs from the heated surface and either shakesor lick the affected appendage. The temperature of the heated surface ispre-set to the experimentally deduced value of 38° C. and a maximumcutoff value of either 53° C. or 54° C. has been used. Data is expressedeither as absolute temperature in degrees C. or as percentage maximalpossible effect as described by the formula ##EQU2## (54 is substitutedfor 53 in the above formula if it is the maximal value.)

GsAF I Administration:

With minimal restraint, peptide GsAF I was injected intrathecally(i.th.) into young (75-150 gram) male Sprague-Dawley rats (CharlesRivers Laboratories, Wilmington, Mass.) into the spinal subarachnoidspace between lumbar spinous processes L4 and L5 using 10 microliterHamilton syringes equipped with 3/8 inch by 28G needles. Dosing levelswere based upon concentrations deduced from ultraviolet absorbancevalues at 280 nm using an extinction coefficient of 18710. Injectionvolume was 10 microliters. The injection vehicle was saline or 0.1%bovine serum albumin(BSA)/saline. The rats were pretreated with GsAF I30 minutes prior to exposure to heat.

With a 30 minute pretreatment interval, 180 picomoles of GsAF 1 (666nanograms) produced a 70% MPE. Lowering the dose ten-fold (to 18picomoles) resulted in the loss of significant activity. Greaterefficacy at multiple doses could be obtained with one hour pretreatment.With one hour pretreatment, a 180 picomole (66 nanograms) dose of GsAF Iproduced a 91% MPE and 18 picomoles (6.6 nanograms) produced a 24% MPE.No adverse motor effects were evident at these doses. This iscorroborated by the observation that the animals' front paws wereresponsive at sub threshold temperatures and were quickly lifted off thehot surface at the elevated temperatures.

GsAF II Administration:

With minimal restraint, peptide GsAF II was injected intrathecally(i.th.) into young (75-150 gram) male Sprague-Dawley rats (CharlesRivers Laboratories, Wilmington, Mass.) into the spinal subarachnoidspace between lumbar spinous processes L4 and L5 using 10 microliterHamilton syringes equipped with 3/8 inch by 28G needles. Dosing levelswere based upon concentrations deduced from ultraviolet absorbancevalues at 280 nm using an extinction coefficient of 18710. Injectionvolume was 10 microliters. The injection vehicle was saline or 0.1%bovine serum albumin(BSA)/saline. The rats were pretreated with GsAF II15 minutes prior to exposure to heat.

A 100 % MPE was recorded for all animals receiving a 2.33 nanomole (9.27micrograms) dose of GsAF II. When the dose was lowered to 583 picomoles(2.32 micrograms), an average MPE of 59% was recorded. No motorcoordination problems were witnessed at these doses.

Example 5--Analgesic Evaluation--von Frey Threshold

In this test filaments of increasing thickness are applied to the dorsalsurface of a hindlimb until the rat either voluntarily removes theappendage with a forcible escape movement or vocalizes. The thickness ofthe filaments are arbitrarily labelled with a value which can betransformed into a grams force reading according to the followingequation: ##EQU3## Data are expressed either as absolute gram forcevalues or a percentage of the maximum possible effect (MPE) as describedin the following equation: ##EQU4## GsAF I Administration:

When tested 30 minutes post injection, doses of 180 picomoles (666nanograms) and 18 picomoles (66 nanograms) of GsAF I produced 93% and24% MPE's, respectively. Extending the pretreatment period to one hourresulted in a 100% MPE for the 180 picomole dose and a 22% MPE for the18 picomole (6.6 nanograms) dose. No confounding side effects werepresent.

GsAF II Administration:

When tested 30 minutes post injection, a 2.33 nanomole (9.27 micrograms)dose of GsAF II produced an MPE of 83%. A 583 picomole (2.32 micrograms)dose of GsAF II produced an MPE of 48%. At the higher dose, 75% of therats tested demonstrated maximal analgesic activity (i.e. 100% MPE).Based upon the GsAF I data, it is assumed that greater analgesicefficacy with GsAF II would be obtained if longer pretreatment wereused.

Example 6--Analgesic Evaluation--Formalin Pain Test

The noxious stimulus for this test is the sub-cutaneous injection of a5% solution of formalin into the dorsal surface of one of the hindlimbsof the animal. Motor activity indices used in this test are 1) the totaltime spent licking that appendage and 2) the total number offlinching/shaking responses of the affected appendage. Data collectionis initiated immediately upon injection of the formalin solution intothe limb. The acute phase response is defined by the time interval of0-5 minutes post formalin injection. The tonic phase response is definedby the interval of 20-35 minutes post formalin injection Data collectionis done in a computerized format. Expression of the data is done usingeither absolute values or as percent control which is defined by thelevel of response following injection of saline vehicle.

GsAF I Administration:

With minimal restraint, peptide GsAF I was injected intrathecally(i.th.) into young (75-150 gram) male Sprague-Dawley rats (CharlesRivers Laboratories, Wilmington, Mass.) into the spinal subarachnoidspace between lumbar spinous processes L4 and L5 using 10 microliterHamilton syringes equipped with 3/8 inch by 28G needles. Dosing levelswere based upon concentrations deduced from ultraviolet absorbancevalues at 280 nm using an extinction coefficient of 18710. Injectionvolume was 10 microliters. The injection vehicle was saline or 0.1%bovine serum albumin(BSA)/saline. The rats were pretreated with GsAF I30 minutes prior to injection with the formalin solution.

The following results were obtained 30 minutes post injection of 180picomoles (666 nanograms) 18 picomoles (66 nanograms) and 1.8 picomoles(6.6 nanograms) of GsAF I. Values are presented as % control withabsolute levels in parentheses.

    ______________________________________           Acute Flinches                     Tonic Flinches                                 Tonic Licking    ______________________________________    Control  (42.5)      (139)       (181.8 sec)    180 pmoles             8.9% (3.8)  3.0% (4.3)  0% (0 sec.)     18 pmoles             20% (8.5)   21% (29.5)  8.4% (15.3 sec)     1.8 pmoles             28% (12)    32% (44.3)  46% (84.3 sec)    ______________________________________

GsAF II Administration:

With minimal restraint, peptide GsAF II was injected intrathecally(i.th.) into young (75-150 gram) male Sprague-Dawley rats (CharlesRivers Laboratories, Wilmington, Mass.) into the spinal subarachnoidspace between lumbar spinous processes L4 and L5 using 10 microliterHamilton syringes equipped with 3/8 inch by 28G needles. Dosing levelswere based upon concentrations deduced from ultraviolet absorbancevalues at 280 nm as stated previously. Based upon a mass of 3980 Da, 583pmoles corresponds to 2.3 micrograms of GsAF II, and 2.33 nmolescorresponds to 9.3 micrograms of GsAF II. Injection volume was 10microliters. The injection vehicle was saline or 0.1 % bovine serumalbumin(BSA)/saline. The rats were pretreated with GsAF II 15 minutesprior to injection with formalin.

    ______________________________________            Acute Flinches                      Tonic Flinches                                 Tonic Licking    ______________________________________    Vehicle Control              (16.8)      (86)       (95.0 sec)    2.33 nmoles              19% (3.25)  1.4% (1.2) 0% (0 sec)     583 pmoles              41% (6.8)   9.5% (8.2) 8.2% (7.8 sec)    ______________________________________

In addition to the above analyses, 180 pmoles (666 nanograms) of GsAF Iwas administered 5 minutes after injection of 5% formalin and the tonicphase responses were recorded as described previously. The highlyefficacious activity of GsAF I was retained. Tonic flinch response wasinhibited 86% (i.e. 14% of control) and tonic lick duration was reduced91 % (i.e. 9% of control). This property has only been reported forstrong analgesic compounds that interact with μ-opioid receptors. Italso demonstrates that the analgesic activity of GsAF I is not dependentupon the interruption of the initial rapid firing of sensory fibers(primarily c-fibers) or occlusion of wind-up within dorsal horn neurons.

Example 7 --Analgesic Evaluation--Opioid Receptor Testing

In order to determine if the anti-nociceptive effects of GsAF I and IIare mediated by opioid receptors, young (75-150 gram) maleSprague-Dawley rats (Charles Rivers Laboratories, Wilmington, Mass.)were pretreated with opioid antagonists at doses that reversed theanti-nociceptive activity of morphine. Animals were then tested in thetail flick test (Example 3) and von Frey tests (Example 5). Subcutaneousadministration of 10mg/kg naloxone 5 minutes prior to the intrathecaladministration of 180 picomoles (666 nanograms) GsAF I failed to inhibitthe analgesic activity of GsAF I (180 pmoles i.th.) measured 10 minuteslater (i.e. 10 minutes post i.th. administration of GsAF I and 15minutes post subcutaneous administration of naloxone), but completelyantagonized the analgesic effect of morphine (3 μg i.th.). Additionally,intrathecal administration of naloxone (50 μg) immediately prior tointrathecal dosing of 180 pmoles GsAF I failed to inhibit analgesicresponse measured 10 minutes later whereas reversal of intrathecalmorphine (3 μg) was determined.

Similarly, pretreatment with the irreversible opioid antagonistB-funaltrexamine (5 micrograms administered intrathecally 18 hours priorto the injection of GsAF I) failed to inhibit the analgesic activity ofGsAF I in either the hot plate test (Example 4) or formalin test(Example 6). The analgesic profile for GsAF I, and presumably GsAF II,indicates high efficacy mediated by a non-opioid receptor relatedmechanism.

Example 8 --Analgesic Evaluation for Cross Tolerance of GsAF I withMorphine

Repeated administration of morphine to both humans and rodents resultsin a decreased analgesic response for any individual dose or a leftwardshift in the dose response curve. This phenomenon, termed tolerance,leads to an escalation of morphine dose for maintenance of equivalentpain relief over time. Clinically, the difference in tolerancedevelopment for analgesia versus negative side effects (i.e. sedation,constipation, respiratory depression, etc) can limit the utility ofmorphine in a chronic treatment regime. Although the physiological basisof tolerance is not completely understood, putative analgesic compoundscan be evaluated to determine if their efficacy is altered as a resultof morphine administration (i.e. morphine cross tolerance). Young(75-150 gram) male Sprague Dawley rats (Charles Rivers Laboratory,Wilmington, Mass.) were subcutaneously dosed twice daily for 6 days withescalating quantities (i.e. 2.5 mg/kg/dose--day 1; 5 mg/kg/dose--day 2;10 mg/kg/dose--day 3; 20 mg/kg/dose--days 4 and 5; 25 mg/kg/dose--day 6)of morphine or with saline as a vehicle control. On the seventh day, theanalgesic activity of morphine and GsAF I was tested using the tonicflinch response of the formalin pain test (as described in Example 6).Dose response determinations for morphine and GsAF I were done usingintrathecal administration into young (75-150 gram) male Spraque Dawleyrats (Charles River Laboratories, Wilmington, Mass.). With minimalrestraint, either morphine or GsAF I was injected into the spinalsubarachnoid space between lumbar spinous processes L4 and L5 using 10microliter Hamilton syringes equipped with 3/8 inch by 28G needles.Dosing levels of GsAF I were based upon concentrations deduced fromultraviolet absorbance values at 280 nm using an extinction coefficientof 18710. Injection volumes were 10 ul for both morphine and GsAF I.Vehicle controls for morphine and GsAF I were saline and 0.1%BSA/saline, respectively.

Animals treated with morphine for 6 days and then evaluated foranalgesia induction as a result of morphine administration on theseventh day exhibited a significant leftward shift (300×) in the doseresponse curve versus animals receiving the saline vehicle (ED₅₀ =0.1 ugi.th. for saline control group; ED₅₀ >30 ug i.th. for morphine treatmentgroup). ED₅₀ is the dose required to give 50% of the maximal analgesiceffect. In contrast, the dose response properties of GsAF I were notsignificantly different for the two treatment arms of the study (ED₅₀--26.7 pmoles i.th. for saline control group; ED₅₀ --20 pmoles i.th. formorphine treatment group) indicating that prior exposure to morphinedoes not alter the analgesic properties of GsAF I or produce crosstolerance.

Example 9 --Analgesia Evaluation--Acute Inflammatory Pain Testing

Tissue injury results in inflammation and hyperalgesia (i.e. increasedmagnitude or duration of pain response to supra threshold noxiousstimuli) at both the site of injury and at adjacent tissue sites. Toassess the anti-hyperalgesic activity of GsAF II in inflammatory painconditions, paw withdrawal latencies were determined in adult 350-400gram male Sprague Dawley rats following the unilateral injection ofcarrageenan, a seaweed extract, into the hindpaw in accordance with themethod of Hargreaves et al, Pain, 32:77-88, 1988. Briefly, withdrawallatencies are measured by placing the rat on a glass plate and focusingradiant heat from the underside of the plate toward the hindpaw surface.Latencies values are recorded in seconds to withdrawal of the hindpawfrom the surface of the plate. Basal measurements are made prior toinjection of the carrageenan (4 mg/hindpaw), followed by a measurementat 150 min post carrageenan injection to obtain the level ofhyperalgesic response. Subsequent to the second measurement, GsAF II orvehicle (i.e. 0:1% BSA/saline) is administered through an indwellingintrathecal cannula positioned within the lumbar enlargement of thespinal cord. Anti-hyperalgesic activity is determined by measuring pawwithdrawal latencies at various time intervals following compoundadministration. Concurrent with the paw withdrawal latencies, physicalmeasurements of paw volume and paw temperature are recorded to detectanti-inflammatory and anti-pyretic activities.

    ______________________________________    Mean Latency Time In Seconds ± SEM                  Vehicle Control                             3 nmol GsAFII                  (n = 8)    (n = 4)    ______________________________________    Base latency prior to                    10.12 ± 0.05                                 8.3 ± 1.2    carrageenan injection    150 minutes post carrageenan                    2.14 ± 0.24                                  3.0 ± 0.81    injection    30 minutes post treatment                    2.73 ± 0.08                                 17.82 ± 3.78    60 minutes post treatment                    3.61 ± 0.40                                 20.8 ± 0.00    ______________________________________

Development of hyperalgesia was detected in all animals tested. As shownin the table above, paw withdrawal times were significantly reduced at150 min post injection of carrageenan. Administration of 3 nmoles ofGsAF II completely reversed this hyperalgesic, thermal-induced responseby increasing paw withdrawal latencies to near maximal values(17.82±3.78 sec) at 30 min and to a predetermined cut-off value (20.8sec) at 60 min. No significant reduction in either paw volume or pawtemperature was detected and there was no indication of confoundingmotor deficits or presence of overt negative side effects. GsAF II is aneffective analgesic/anti-hyperalgesic compound for acute peripheralinflammatory pain. GsAF II does not appear to possess anti-inflammatoryproperties since the induction of analgesia was not associated with anacute reduction of edema or of body temperature.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 2    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 29 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 29    (D) OTHER INFORMATION: /note= "Xaa is amidated leucine"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    TyrCysGlnLysTrpLeuTrpThrCysAspSerGluArgLysCysCys    151015    GluAspMetValCysArgLeuTrpCysLysLysArgXaa    2025    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 31 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    TyrCysGlnLysTrpMetTrpThrCysAspGluGluArgLysCysCys    151015    GluGlyLeuValCysArgLeuTrpCysLysLysLysIleGluTrp    202530    __________________________________________________________________________

I claim:
 1. A method of identifying compounds that mimic the analgesiaactivity of a peptide having the amino acid sequence of SEQ ID NO: 1 orSEQ ID NO: 2, comprising the steps of:a) conducting a biological assayon a peptide having the amino acid sequence of SEQ ID NO:1 or SEQ IDNO:2 to determine the analgesia activity, b) conducting a biologicalassay on a test compound to determine the analgesia activity; and, c)comparing the results obtained from the biological assay of SEQ ID NO: 1or SEQ ID NO: 2 to the results obtained from the biological assay of thetest compound.
 2. The method of claim 1 wherein said peptide is thepeptide having the amino acid sequence of SEQ ID NO:
 1. 3. The method ofclaim 1 wherein said peptide is the peptide having the amino acidsequence of SEQ ID NO:
 2. 4. A method of identifying compounds thatmimic the analgesia activity of a peptide having the amino acid sequenceof SEQ ID NO: 1 or SEQ ID NO: 2 , comprising the steps of:a) contactinga labeled peptide having the amino acid sequence of SEQ ID NO: 1 or SEQID NO: 2 with a sample, b) adding a test compound to the sample incontact with a labeled peptide having the amino acid sequence of SEQ IDNO: 1 or SEQ ID NO: 2; and, c) measuring the binding of a labeledpeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2with the sample.
 5. The method of claim 4 wherein said peptide is thepeptide having the amino acid sequence of SEQ ID NO:
 1. 6. The method ofclaim 4 wherein said peptide is the peptide having the amino acidsequence of SEQ ID NO: 2.